This invention relates generally to the field of miniaturized devices for conducting chemical and biochemical processes, and more particularly relates to a novel microreactor for conducting DNA amplification using the polymerase chain reaction, or xe2x80x9cPCR.xe2x80x9d
In sample analysis instrumentation, smaller dimensions generally result in improved performance characteristics and at the same time result in reduced production and analysis costs. Miniaturized separation systems, for example, provide more effective system design, result in lower overhead, and enable increased speed of analysis, decreased sample and solvent consumption and the possibility of increased detection efficiency.
Accordingly, several approaches have been developed in connection with miniaturization of devices for use in chemical analysis, particularly in micro-column liquid chromatography (xcexcLC), wherein columns with diameters of 100 to 200 microns are used, in capillary electrophoresis (CE), wherein electrophoretic separation is conducted in capillaries on the order of 25 to 100 microns in diameter, and in microchannel electrophoresis (MCE), wherein electrophoresis is carried out within a microchannel on a substantially planar substrate. The conventional approach in miniaturization technology as applied to CE and xcexcLC involves use of a silicon-containing material, i.e., a capillary fabricated from fused silica, quartz or glass. With MCE, an attractive method that is useful in conjunction with high throughput applications and enables reduction in overall system size relative to CE, miniaturized devices have been fabricated by silicon micromachining or lithographic techniques, e.g., microlithography, molding and etching. See, for example, Fan et al. (1994) Anal. Chem. 66(1):177-184; Manz et al., (1993) Adv. in Chrom. 33:1-66; Harrison et al. (1993), Sens. Actuators, B B10(2):107-116; Manz et at. (1991), Trends Anal. Chem. 10(5):144-149; and Manz et at. (1990) Sensors and Actuators B (Chemical) B1(1-6):249-255. The use of micromachining techniques to fabricate miniaturized separation systems in silicon provides the practical benefit of enabling mass production of such systems, and there are a number of techniques that have now been developed by the microelectronics industry for fabricating microstructures from silicon substrates. Examples of such micromachining techniques to produce miniaturized separation devices on silicon or borosilicate glass chips can be found in U.S. Pat. Nos. 5,194,133 to Clark et al., 5,132,012 to Miura et al., 4,908,112 to Pace, and 4,891,120 to Sethi et al.
Use of silicon-containing substrates such as fused silica, quartz and glass in microanalytical devices is problematic in a number of ways. For example, silicon dioxide substrates have high energy surfaces and strongly adsorb many compounds, most notably bases. Silicon dioxide materials also dissolve to an appreciable extent when used with basic solutions. Furthermore, when used in electrophoretic applications, the internal surface of a silica capillary or microchannel will be negatively charged at basic pH as a result of deprotonation of surface silanol groups (i.e., they are in the form of anionic, Sixe2x80x94Oxe2x88x92, groups). The surface charge on the interior of the capillary or microchannel not only exacerbates the problem of unwanted adsorption of solute, but also modulates the velocity of electroosmotic flow (also termed xe2x80x9celectroendoosmotic flowxe2x80x9d or EOF) on an unmodified surface, in turn affecting the sensitivity and reproducibility of the chemical analysis conducted. (That is, the EOF velocity is a function of zeta potential xcex6, which is essentially determined by surface charge.) Microfabrication using silicon per se is similarly problematic insofar as a silica surface will form on a silicon substrate under even mildly oxidizing conditions.
For the foregoing reasons it would be desirable to fabricate microanalytical and other miniaturized devices from materials that are not silicon-based, e.g., using inexpensive and readily available polymeric materials. It would also be desirable to extend the utility of microdevices beyond electrophoretic and chromatographic separation techniques to other types of chemical processes, processes that may involve high temperatures, extremes of pH, harsh reagents, or the like. The present invention provides such microanalytical devices.
The area with which the present invention is concerned is in bioanalysis. An important technique currently used in bioanalysis and in the emerging field of genomics is the polymerase chain reaction (PCR) amplification of DNA. As a result of this powerful tool, it is possible to start with otherwise undetectable amounts of DNA and create ample amounts of the material for subsequent analysis. The technique is described in U.S. Pat. No. 4,683,195 to Mullis et al. and related U.S. Pat. Nos. 4,683,202, 4,800,159 and 4,965,188 to Mullis et al. Automated systems for performing PCR are known, as described, for example, in U.S. Pat. Nos. 5,333,675 and 5,656,493 to Mullis et al. PCR uses a repetitive series of steps to create copies of polynucleotide sequences located between two initiating (xe2x80x9cprimerxe2x80x9d) sequences. Starting with a template, two primer sequences (usually about 15-30 nucleotides in length), PCR buffer, free deoxynucloside tri-phosphates (dNTPs), and thermostable DNA polymerase (commonly Taq polymerase), one mixes these components, and then heats to separate the double-stranded DNA. A subsequent cooling step allows the primers to anneal to complementary sequences on single-stranded DNA molecules containing the sequence to be amplified. Replication of the target sequence is then accomplished by the DNA polymerase which produces a strand of DNA that is complementary to the template. Repetition of this process doubles the number of copies of the sequence of interest, and multiple cycles increase the number of copies exponentially.
Since PCR requires repeated cycling between higher and lower temperatures, PCR devices must be fabricated from materials capable of withstanding such temperature changes. The materials must be mechanically and chemically stable at high temperatures, and capable of withstanding repeated temperature changes without mechanical degradation. Furthermore, the materials must be compatible with the PCR reaction itself, and not inhibit the polymerase or bind DNA.
To date, however, there remain many problems with performing PCR in microdevices. One problem involves the low thermal stability of many materials. That is, many types of materials, e.g., polymeric materials, cannot withstand the cycling temperatures used in PCR, typically in the range of about 37xc2x0 C. to 90xc2x0 C., without significant or complete loss of mechanical integrity. In addition, contaminants may be present on or leach out of a substrate surface, affecting the precise balance of appropriate ingredients (metal ions, salts, buffering systems, oligonucleotides, primers, and polymerases) required for PCR, in turn resulting in unsuccessful amplification reactions. Also, the polymerase enzyme or any of the components involved in the PCR reaction may bind to or become adsorbed on a microchannel surface. Contact between the polymerase and a substrate surface will generally result in irreversible denaturation. These types of xe2x80x9cbiofoulingxe2x80x9d are especially problematic with capillaries or microchannels of micron or submicron dimensions because of the very high surface area to volume ratio.
The present invention addresses the aforementioned needs in the art, and provides a PCR microreactor for amplifying DNA using microquantities of sample fluid. In its simplest embodiment, the PCR microreactor comprises: a reaction chamber defined by two or more interior surfaces, a means for introducing PCR reaction components into the chamber, a means for removing the PCR reaction product from the chamber, and a means for controlling the temperature of the reaction chamber,
wherein the device is fabricated from a material that is thermally, chemically and mechanically stable under the conditions at which a PCR reaction is conducted, and employs a reaction chamber adapted to contain in the range of approximately 1 xcexcl to 500 xcexcl of fluid. Preferred materials are those that exhibit reduced adsorption of solute, e.g., biomolecules such as proteins, nucleic acids, etc., and can be modified, coated or otherwise treated so as to optimize electroosmotic flow.
A second embodiment of the invention the PCR microreactor comprises:
a substrate having first and second substantially planar opposing surfaces, with a cavity and at least one microchannel formed in the first planar surface, wherein the cavity serves as a reaction zone that is in fluid communication with each microchannel;
a cover plate arranged over the first planar surface, which in combination with the cavity defines a reaction chamber, and with each microchannel defining a microcolumn; and
at least one inlet port and at least one outlet port communicating directly or indirectly with the reaction chamber, enabling the passage of fluid from an external source into and through the reaction chamber,
wherein the substrate and the cover plate are comprised of a material that is thermally and chemically stable and resistant to biofouling.
An additional embodiment of the invention presents a method for conducting the polymerase chain reaction (PCR) to amplify DNA in a sample which comprises heating the sample to separate double-stranded DNA into single-stranded DNA, cooling the sample so as to allow hybridization of primer oligonucleotides to the single-stranded DNA, replicating the DNA using a DNA polymerase, and repeating the aforementioned steps to achieve the desired degree of amplification,
wherein the PCR is conducted in a microreactor comprised of a material that is thermally, chemically and mechanically stable under the conditions at which the PCR reaction is conducted, and employs a reaction chamber adapted to contain approximately 1 xcexcl to 500 xcexcl of fluid.
In a further embodiment a method is presented for amplifying the quantity of a DNA molecule of interest contained in a small volume of sample fluid using the polymerase chain reaction, comprising:
(a) introducing into a microreactor up to about 10 xcexcl of a sample fluid containing the DNA molecule of interest in double-stranded form, a first and a second primer molecule complementary to opposing strands of the DNA molecule, a thermostable DNA polymerase, free deoxynucleoside triphosphates and PCR buffer, the microreactor comprising
a substrate having first and second substantially planar opposing surfaces, said substrate having a cavity formed in the first planar surface, wherein the cavity serves as a reaction zone,
a cover plate arranged over the first planar surface, said cover plate in combination with the cavity defining a reaction chamber, and
at least one inlet port and at least one outlet port in fluid communication with the reaction chamber, said ports enabling the passage of sample fluid from an external source into and through the reaction chamber, thereby defining a fluid flow path,
wherein the substrate and the cover plate are comprised of a material that is thermally stable and resistant to biofouling;
(b) applying a motive force to the device to move the sample fluid along the flow path into the reaction chamber;
(c) heating the sample fluid in the reaction chamber to separate the double-stranded DNA into single-stranded DNA;
(d) cooling the sample so as to allow hybridization of the primer molecules to opposing strands of the single-stranded DNA and replication of the single-stranded DNA by the DNA polymerase; and
(e) repeating steps (c) and (d) to achieve the desired degree of amplification.
In a still further embodiment of the invention, a method is presented for amplifying the quantity of a DNA molecule of interest contained in a small volume of sample fluid using the polymerase chain reaction, comprising:
(a) introducing into the microreactor of the second embodiment of the invention up to bout 10 xcexcl of a sample fluid containing the DNA molecule of interest in double-stranded form, first and a second primer molecule complementary to opposing strands of the DNA molecule, a hermostable DNA polymerase, free deoxynucleoside triphosphates and PCR buffer,
(b) applying a motive force to the device to move the sample fluid along the flow path into the reaction chamber;
(c) heating the sample fluid in the reaction chamber to separate the double-stranded DNA to single-stranded DNA;
(d) cooling the sample so as to allow hybridization of the primer molecules to opposing strands of the single-stranded DNA and replication of the single-stranded DNA by the DNA polymerase; and
(e) repeating steps (c) and (d) to achieve the desired degree of amplification.